Pyridoxal 5'phosphate (PLP) is an essential cofactor that catalyzes a wide range of reactions involving amines and amino acids. E. coli and other 3- proteobacteria synthesize PLP from erythrose 4-phosphate and 1-deoxy-D- xylulose 5-phosphate. A strain lacking PdxB (erythronate 4-phosphate dehydrogenase) cannot grow on glucose because it cannot make PLP. We have found that overexpression of seven different enzymes allows this strain to grow slowly on glucose. Two of these (PdxA and AroB) probably have promiscuous PdxB activity. The remaining five appear to facilitate one of two different latent pathways that allow the step blocked by the absence of PdxB to be bypassed. The first of these pathways appears to be patched together using three enzymes that normally serve other functions and a protein of unknown function. The enzymes involved in the second pathway have not yet been identified. This project will characterize the suspected promiscuous activities of PdxA and AroB and the enzymes involved in both latent pathways. We will use genome shuffling to evolve strains of E. coli that use the latent PLP synthesis pathways more efficiently. We will characterize the evolved strains by genome re-sequencing, transcriptional profiling, and various biochemical approaches to identify the mechanisms by which the strains have adapted to use a latent pathway more efficiently. This project is novel because it addresses the evolutionary potential of "roads not taken". While it is obvious that nature has not explored all possible solutions to the synthesis of critical metabolites, we rarely have an opportunity to explore the potential of a pathway that might serve as well as those found in extant organisms. Our analysis of the genetic changes required for adaptation to the use of the inefficient latent pathways for PLP synthesis will inform other efforts to incorporate novel metabolic modules into the pre-existing metabolic network of E. coli and other bacteria for industrial purposes. In addition, this project will enhance our understanding of the potential for assembling novel metabolic pathways by patching together enzymes that normally serve other functions in the cell. Such pathways could allow degradation of anthropogenic chemicals such as antibiotics, pesticides, and industrial pollutants.